Need a paired-end RNA-Seq library construction protocol

[satp]

Hi all,

I want to construct an RNA-Seq library used for paired-end sequencing using HiSeq2000 and need a protocol. I have found many version of modified protocol in PubMed, such as construction of strand-specified RNA-seq library. I also found some protocol of RNA-Seq library preparation kits, but I don't want to used these kit and the protocol is not detailed enough.

Is there anyone have such protocol or know where I can get it?

Jian-You

[mcnelson.phd]

Hi Jian-You,

I've been using the strand specific protocol developed by Parkhomchuck et al. and have very good success with it. Essentially, you make cDNA, with the second strand synthesis incorporating UTP instead of TTP. Shear your cDNA to your desired size, then run it through the Illumina TruSeq DNA prep method. Right before the final PCR step you treat your library with USER from NEB to enforce the strand specificity.

Regardless of which method you ultimately choose, you'll likely have to use some kit in order to get the Illumina adapters incorporated.